Differentiation and proliferation of hemtopoietic cells are regulated by glycoproteins referred to as colony stimulating factors (CSFs). Of the various CSFs, the Granulocyte Colony Stimulating Factor (GCSF) stimulates the proliferation of specific bone marrow precursor cells and their differentiation into granulocytes. When administered to mammals, GCSF promotes a dramatic increase in circulating granulocyte populations.
GCSF is one of several proteins produced by recombinant DNA technology for therapeutic use. Of the two types of GCSF clinically available, lenograstim, the glycosylated form of GCSF, is expressed in mammalian cells, and filgrastim, the non-glycosylated form is expressed in Escherichia coli (E. coli).
Proteins expressed by recombinant DNA methods in bacteria such as E. coli, are usually expressed as insoluble aggregates called inclusion bodies. These protein aggregates are separated, solubilized in the presence of denaturing agents. Solubilized protein is then refolded in presence of oxidizing agents. The refolded protein can then be further purified by appropriate column chromatographic procedures.
A typical production process for GCSF is described in U.S. Pat. No. 5,849,883. Recombinant GCSF is obtained by lysing E. coli cells and separating out the inclusion bodies containing GCSF. Inclusion bodies are then solubilized with deoxycholate and the extracted GCSF is refolded in the presence of a denaturant agent and an oxidizing agent. Refolded protein is further purified using two ion exchange chromatography steps.
In addition to U.S. Pat. No. 5,849,883, other prior art also disclose various methods for refolding of proteins. EP0547102 explains a process of solubilization of proteins by addition of cationic surfactant and providing conditions to allow for refolding.
EP1630173 teaches a method for solubilization of GCSF from inclusion bodies by using reduced glutathione and a denaturing agent for solubilizing inclusion bodies.
U.S. Pat. No. 7,538,198 and EP1434789 provide a process of refolding of a protein by subjecting a protein aggregate and a denaturing agent to increased pressure and removing the dissolved protein from the increased pressure to fold the protein.
US2011294990 explains a process of refolding a protein by using two chaotropic agents and US2011034678 describes a process of refolding of a protein by using guanidine in the refolding buffer.
WO2011113601 teaches a process for refolding of GCSF by using oxidized glutathione and at least one reversed phase chromatography.
GCSF, obtained by solubilization and refolding of inclusion bodies, contains the native, oxidized and the reduced forms of GCSF. The reduced forms of GCSF are reported to form aggregates, whereas the oxidized form of GCSF have reduced bioactivity (Reubsaet et al., J. Pharm. Biomed. Anal. 17, 283-289, 1998). Generally, a chromatographic step is used downstream of refolding which caters to removal of the oxidized impurities generated during solubilization and refolding.
Refolding of GCSF by methods as discussed in the above stated prior art, results in higher presence of oxidized impurities resulting in sub-optimal yields of the correctly folded biologically active protein. Methods to reduce the creation of these oxidized impurities during solubilization would circumvent the need for an additional step to remove oxidized impurities. The current invention provides a method to reduce the generation of oxidized impurities by an optimized solubilization process during refolding of GCSF.